Journal of Colloid and Interface Science 453 (2015) 202–208
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Surface chemistry and spectroscopy of the b-galactosidase Langmuir monolayer Nicholas F. Crawford a, Miodrag Micic b,c, Jhony Orbulescu b, Daniel Weissbart d, Roger M. Leblanc a,⇑ a
Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, United States MP Biomedicals LLC, 3 Hutton Center, Santa Ana, CA 92707, United States c Department of Engineering Design Technology, Cerritos College, 11110 Alondra Boulevard, Norwalk, CA 92650, United States d MP Biomedicals SAS, Parc d’innovation-Rue Geiler de Kaysersberg, Illkirch-Graffenstaden 67402, France b
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 25 February 2015 Accepted 29 April 2015 Available online 7 May 2015 Keywords: b-Galactosidase Langmuir monolayer Spectroscopy Photophysical properties
a b s t r a c t The changes of interfacial properties of b-galactosidase introduced into different pH environments are investigated through surface chemistry and in situ spectroscopy. Conditions for an optimal Langmuir monolayer formation were firstly obtained by varying the subphase salt concentration and the surface-pressure area isotherm was used to extrapolate the limiting molecular area of the enzyme monolayer to be around 42,000 Å2 molecule 1. Surface pressure stability measurements held at 20 mN/m for 90 min along with compression–decompression cycles revealed no aggregate formation at the air–water interface. Consistent with the data obtained from the isotherm, in situ UV–Vis and fluorescence spectroscopy shows a steep rise in absorbance and photoluminescence intensity correlating to with a switch from a liquid-expanded to a liquid-condensed phase. A decrease in subphase pH increased the electrostatic repulsion as the enzyme was protonated, leading to an expanded monolayer. Infrared absorption–reflection spectroscopy demonstrates that the enzyme adopts mainly b-sheet conformation at the air–water interface before and during the compression. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
⇑ Corresponding author. E-mail address: [email protected] (R.M. Leblanc). http://dx.doi.org/10.1016/j.jcis.2015.04.063 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
b-Galactosidase (E.C. 3.2.1.23) is an important family of hydrolase exoglycosidase enzymes [1,2]. Its primary function is to cleave b-glycosidic bond formed between a galactose and organic
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substrate [3,4]. To a lesser extent, this enzyme can also act on the arabinosidic and fucosidic bonds with other organic residues, such as amino acids, and it is an essential enzyme for the metabolism of both eukaryotic and prokaryotic cells. Besides fundamental interests in its role in metabolism, b-galactosidase is important tool in biochemistry, life science research and industrial biotechnology [5,6]. Its action on the X-gal substrate results in cleaving the glycosidic linkage to the indigo type dye, creating a strong indigo blue color which is a base reporting system for many ELISA assays as well as for histochemical staining [7,8]. It is also important during gene expression as a reporter gene and is the basis of the blue– white screen in molecular cloning experiments. Furthermore, its large emerging applications are in the field of glycomics, where it is used extensively in sample preparation for glycan cleavage [9]. In this paper we explore the biophysical properties of b-galactosidase at the air–water interface. A large biomacromolecule such as b-galactosidase is interesting not only for its role in biological systems as a hydrolyzing enzyme but also for its behavioral characterization which has been previously uncharacterized in a two-dimensional environment [10,11]. The Langmuir monolayer approach has been used extensively in the past to study proteins [12–14], lipids [15,16], polymers [13,17] and nanoparticles [18,19] at the interfaces, as well as to understand their structural behaviors with different environmental conditions in two dimensions. Langmuir monolayer technique has applications of simulating a hydrophobic–hydrophilic cellulous environment to investigate biomolecules at the interfaces [20,21]. The major advantages of this technique are the ease of controlling both the intermolecular interaction and ordering of the analyte via controllable variables, such as subphase and monolayer component, pH, surface pressure, surface potential and temperature [22]. Among the advantages, an important parameter easily controlled is the surface pressure that is exerted by the monolayer. When simulating a biological membrane higher surface pressures are required to accurately analyze the state of the analyte as it is compressed from an expanded phase with little intermolecular interaction to a condensed phase where high amounts of intermolecular interactions and changes in orientation are observed [23,24]. Surface-pressure area (p–A) isotherms are an ideal method of analyzing the compression of a monolayer formed by molecules such as b-galactosidase as it reduces the surface tension of the liquid subphase contained in a Langmuir trough system. Once a molecule is deposited at the air–water interface, immediate repulsion– expansion effects occur and the force energy per unit area of the subphase is lowered. As barriers skim the surface of the subphase, compressing the molecules closer together and reducing their orientation degrees of freedom, reorganization and reorientation stabilize the monolayer [23]. The force per unit length is then represented as a positive difference between the surface tension of the subphase, which should not increase in surface pressure during compression, and the increase in surface tension of the b-galactosidase monolayer until the monolayer collapses [25]. In addition to measurements involving surface-pressure, surface-potential area isotherms are performed which detail the difference in potential between the molecules at the interface and a vibrating electrode. In conjunction, these two methods reveal the state of the molecules starting at points of zero interaction and compressing them to form a condensed film and finally a compact monolayer. The heterogeneity of the monolayer is described by comparing the two isotherms which show at certain points of compression, the molecules exhibit a greater degree of electrostatic interaction between themselves and also with the subphase. Those interactions too minute to observe with physical movements such as dipole moment changes and van der Waals forces can therefore be detected.
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Not only are the physical interactions observable but also in the case of Langmuir monolayers, the photophysical properties can be determined as the molecules move to a restricted packing structure at the interface [26,27]. b-Galactosidase has a high content of tryptophan and tyrosine making it ideal to study by utilizing two in situ spectroscopic methods: UV–Vis and fluorescence spectroscopy. The high tryptophan and tyrosine content of b-galactosidase is expected to absorb incident light and produce an intensity of emission throughout these experiments [28,29]. In this article, the Langmuir monolayer technique has been implemented to analyze the surface chemistry and in situ spectroscopy of b-galactosidase during monolayer compression. Experimentation of the stability has revealed the influence of pH on the nature of the enzyme while at the air–water interface.
2. Materials and methods 2.1. Materials b-galactosidase was obtained from MP Biomedical (Solon, OH) with a monomer molecular weight of 116,646 Da as determined by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometer. The specific activity of the enzyme (735 units/mg dry weight) was provided by the company by analysis with o-nitrophenyl-b-D-galactopyranoside (ONPG) which defines one unit of the enzyme which hydrolyzes 1 lmole of the substrate per minute at 25 °C, pH 7.5. Hydrochloric acid and sodium chloride with a purity higher than 99.5%, were also obtained from MP Biomedical. Sodium hydroxide used for adjusting pH was obtained from Pharmco (Brookfield, CT). All chemicals were used without any further purification. Water utilized in these experiments was obtained from a Modulab 2020 Water purification system (Continental Water System Corp. San Antonio, TX) with resistivity of 18 MX cm, surface tension of 71.6 mN/m, and pH 5.7 at 20.0 ± 0.5 °C.
2.2. Methods Langmuir isotherms and in situ UV–Vis absorption and fluorescence spectroscopy were conducted in a clean room (class 1000) with constant temperature of 20.0 ± 0.5 °C and humidity of 50 ± 1%. A Kibron l-trough (Kibron Inc., Helsinki, Finland) with an area of 5.9 cm 21.1 cm was utilized for the surface pressure– and surface potential–area isotherms, compression–decompres sion cycles, and stability studies. The Wilhelmy method was utilized to measure surface pressure with a 0.51 mm diameter alloy wire probe with a sensitivity of ±0.01 mN/m. Surface potential was measured with a Kelvin probe consisting of a capacitor-like system. The vibrating plate was set at 1 mm above the surface of the Langmuir monolayer and a gold-plated trough acted as a counter electrode. UV–Vis absorption implemented at the air–water interface of the b-galactosidase Langmuir monolayers were obtained with a Hewlett–Packard 8452A spectrophotometer. Fluorescence spectra for b-galactosidase Langmuir monolayers were measured with an excitation wavelength kexc = 284 nm by an optical fiber detector connected to a Fluorolog-3 spectrofluorometer (Horiba Scientific, Edison, NJ) and excitation/emission both had slit widths set to 3 nm. Analysis was conducted using the maximum emission wavelength of the enzyme produced by the tryptophan moiety, kem = 354 nm. Both UV–Vis absorption and fluorescence spectra were collected on the top of a KSV trough (KSV Instrument Ltd., Helsinki, Finland), which has an area of 7.5 cm 30 cm and a quartz window located in the center of the trough. Photophysical
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3. Results and discussion 3.1. Surface pressure– and surface potential–area isotherms The interfacial properties and Langmuir monolayer behavior of b-galactosidase were investigated to optimize conditions keeping the enzyme at the air–water interface and producing a stable monolayer. Investigations of subphase salt concentration as well as spreading volume resulted in Langmuir monolayer formation with an enzyme concentration of 0.2 mg/mL and a spreading volume of 65 lL on a pH 5.7, 0.1 M NaCl subphase. Conducted in triplicate, these conditions reproducibly demonstrated all necessary phases of a proper formation of a two-dimensional enzyme Langmuir monolayer. Fig. 1 shows combined surface pressure– and surface potential– area isotherms. The surface pressure–area isotherm calculated to a spreading amount of 2 10 11 mol of enzyme at the air–water interface, a small amount which is necessary due to the large molecular weight of the molecule. An initial zero in terms of surface pressure starting at 80,000 Å2 molecule 1 is ideal correlating to a gaseous (G) phase where the floating film at the hydrophobic–hydrophilic interface has little to no intermolecular interactions [31]. Compression of the molecules at the surface results in an increase in surface pressure of the expanded film as described by a decrease in area per molecule. The liquid-expanded (L-E) phase of the monolayer starting to occur at around 65,000 Å2 molecule 1 is of interest because it shows that the molecules’ degrees of freedom are being reduced as their orientation becomes uniform. The enzyme molecules continue to condense until 30,000 Å2 molecule 1 when a steep increase in surface pressure is observed referred to as the liquid-condensed (L-C) phase where the homogenous monolayer is analogous to a three-dimensional
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spectra taken of the b-galactosidase aqueous solutions were measured by a Shimadzu UV2600 spectrophotometer recording spectra between 800 and 200 nm and a Fluorolog-3 spectrofluorometer with a slit width of 3 nm for the excitation/emission spectra, respectively. The infrared absorption–reflection spectroscopy (IRRAS) measurements of the enzyme Langmuir monolayer at the air-aqueous interface were characterized using an EQUINOX 55 Fourier transform infrared spectrometer (Bruker Optics, MA) which was connected with an XA-511 external reflection accessory equipped a mercury–cadmium–telluride (MCT) detector. The studies of IRRAS were performed using p-polarized light on a Kibron l-trough S. Each spectrum was acquired by the co-addition of 1200 scans with a resolution of 8 cm 1. UV–Vis absorption was used to analyze and confirm the concentration of the b-galactosidase bulk solution. The concentration was accurately calculated based on dilutions of the enzyme, initially a dry powder, kept at 4 °C by utilizing the reported E1% (20.9) of the enzyme [30]. Langmuir monolayers were obtained with a bulk solution concentration of 0.2 mg/mL relating to an absorbance of 0.4 at the maximum absorption of tryptophan moiety, kmax = 281 nm. Reproducible monolayers were obtained on a sodium chloride subphase (0.1 M) and the spreading volume of the enzyme was 65 lL while using the Kibron instrument and 150 lL while using the KSV instrument. At the air–water interface, deposition of the enzyme in solution was conducted by placing droplets of equal size on the surface of the subphase using a 100 lL syringe (Hamilton Co., Reno, Nevada). Experiments were then continued after a waiting time period of 15 min in order to allow the b-galactosidase monolayer to reach equilibrium at a compression rate of 6086 Å2 molecule 1 min 1. In a typical surface pressure or potential-area isotherm experiment, the barriers usually reached the maximum compression area.
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Mean Molecular Area ( Å molecule ) Fig. 1. Surface pressure–area and surface potential–area isotherms for 2 10 b-galactosidase spread on a pH 5.7, 0.1 M NaCl subphase.
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liquid. The higher surface pressure regions are of importance because of the high tension, 30–35 mN/m, found in biological membranes where molecules have the least amount of compressibility [23,32]. The final phase observed in a well-ordered monolayer is occurring around 22,000 Å2 molecule 1 where the maximum compression is reached. Extrapolating the surface pressure–area curve during the steepest part of the isotherm, typically during the liquid-condensed phase (dash line in Fig. 1), down to a zero surface pressure allows to determine the limiting molecular area [33]. The Langmuir monolayer formed from b-galactosidase has a limiting molecular area of 42,000 (±3400) Å2 molecule 1. Considering the three-dimensional crystal structure of beta-galactosidase from E. coli. (PDB: 4V40) with 107.90 Å 207.50 Å 509.90 Å, one would expect the enzyme is likely orientated as 107.90 Å 509.90 Å at the air–water interface with the axis of 207.50 Å toward the air phase. Surface potential measures the potential or dipole moment difference above and below the Langmuir monolayer film. In conjunction with surface pressure, surface potential–area isotherms as previously discussed reveal the molecular interactions occurring before and during any type of phase change of the monolayer as seen during compression. These changes in surface-potential can be correlated with phase changes in the monolayer as the minimum cross-sectional area per molecule at the air–water interface decreases. In the case of b-galactosidase, the surface potential is most influenced by dipole moment changes throughout the compression. Fig. 1 correlates the two types of monolayer measurements by showing even during the initial gaseous phase, there is an immediate increase in surface potential at 80,000 Å2 molecule 1. A kink in surface potential is observed as the monolayer begins to move into a liquid-expanded phase and as the monolayer compression transitions into a liquid-condensed phase, again a change in surface potential is observed. At a maximum voltage of 108 mV, the surface potential corresponds to a close packing structure of the enzyme molecules at the beginning of the liquid condensed phase. In the liquid-condensed phase region, while the molecular rearrangement is happening, due to the short distance (surface pressure–area isotherm), some of the dipole–dipole interaction will cancel out therefore resulting in the overall effect that the surface potential in this region is slightly decreasing. When compared, the two isotherm methods seem slightly non-aligned which is explained by the difference in detected dipole changes compared to intermolecular interactions. Surface potential is considered more sensitive in measuring these changes since
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they occur at larger distances compared to the distance needed for intermolecular interaction [34]. 3.2. The compression–decompression cycles and stability measurements of the b-galactosidase Langmuir monolayer The capacity of a monolayer to be stable for long time periods is necessary in order to perform certain in situ experimentation therefore compression–decompression cycles are conducted which
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reveal the extent of analyte which is being lost to desorption from the interface into the subphase, also known as hysteresis [35]. Once a stable and reproducible monolayer was formed, these cycles reveal in Fig. 2A–C that when compressed to 10 mN/m there is a small hysteresis because a stable monolayer has not yet been formed at this surface pressure. Although only 4% is of the initial isotherm is lost upon comparison of the first and third cycle, 20 and 30 mN/m only show hysteresis loss of 1% and 2%, respectively. The possible reason for the smaller hysteresis loss is likely due to the larger rigidity of the Langmuir monolayer when it is compressed to the higher surface pressures. Reorganization of the molecules at the interface shows that under these experimental conditions, the salt subphase renders the b-galactosidase molecules partially insoluble and good reversibility of the isotherm shows that there is little aggregate formation at the interface as well [34,36]. The stability is obtained by compressing the monolayer to the target surface pressure and keeping the surface pressure constant. The monolayer stability is evaluated using the decrease in the surface area attributed to the dissolution of the enzyme in the liquid subphase. The excellent stability of the enzyme monolayer held at a surface pressure of 20 mN/m for over a 90 min time period in Fig. 3A shows an approximate change in area per molecule to be 18%. Ideal for future experimentation, the one-molecule-thick monolayer of b-galactosidase molecules can be analyzed at this point during compression as it is transitioning between the liquid-expanded and liquid-condensed phases. Fig. 3B shows that when holding the monolayer at a higher surface pressure of 30 mN/m, a 20% shift in area per molecule occurs over the first 30 min, after which the monolayer may collapse or lose molecules to the subphase. The instability of the monolayer at this point in compression is evident by the early collapse showing that this surface pressure is not suitable for time dependent measurements. 3.3. Effect of pH on limiting molecular area
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Mean Molecular Area (Å molecule ) Fig. 2. Compression–decompression cycles for the b-galactosidase Langmuir monolayer on a 0.1 M NaCl subphase up to (A) 10, (B) 20 and (C) 30 mN/m. The arrow in each figure shows the first to the third cycle of the compression– decompression.
The limiting molecular area of the b-galactosidase Langmuir monolayer describes the minimum cross-sectional area per molecule. This value can however change with environmental conditions, namely the subphase conditions. As the extrapolated value for the limiting molecular area is shifted due to these changes, the molecular secondary conformation can be analyzed at the air–water interface. Changes in pH can affect the conformation of an enzyme by destabilizing certain interactions between amino acids throughout the molecule. Fig. 4 shows the effect of pH on the b-galactosidase Langmuir monolayer by adjusting the subphase pH while keeping the salt concentration constant at 0.1 M NaCl. The isotherms seem to collapse at the same molecular area, but this ‘‘collapse’’ is not real due to the fact that the barriers are compressed to the maximum. The isotherm initially was obtained on a subphase of pH 5.7 which is close to the isoelectric point of the enzyme, 4.6. When the pH of the subphase is around the isoelectric point of b-galactosidase, the charges resulting from functional groups that comprise the enzyme cancel out to a neutral charge, facilitating the formation of Langmuir monolayer at the air–water interface. Decreasing the pH increases the mean molecular area of the enzyme resulting in a more expanded monolayer upon compression due to a greater degree of electrostatic repulsion. This type of repulsion is expected since the overall negative charge of the enzyme based on the amino acid composition will be protonated therefore instead of the molecules being closely packed, the molecules are more spread out at the interface. On the contrary, increasing the pH away from the isoelectric point reduces the mean molecular area (Fig. 4). This is not an unexpected result from the enzyme monolayer since the basic subphase solubilizes the
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Fig. 3. Stability measurements which shows over an extended time period if the b-galactosidase monolayer can stay at the air–water interface when compressed to higher surface pressures of (A) 20 and (B) 30 mN/m. The double arrows in the figure show the percentage of mean molecular area shift at (A) 90 min and (B) 30 min in comparison with the mean molecular area when the monolayer was initially compressed to 20 and 30 mN/m.
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enzyme and hinders monolayer formation. Studies past a subphase of pH 8 (data not shown) resulted in no isotherm being produced which shows that solubilizing effects of the subphase cause the molecules to be expelled into the subphase before any type of monolayer can be formed. 3.4. In Situ photophysical properties of the b-galactosidase Langmuir monolayer 3.4.1. UV–Vis absorption & fluorescence spectroscopy The UV–Vis absorption and fluorescence spectroscopy of the b-galactosidase Langmuir monolayer were conducted on a 0.1 M NaCl subphase with a larger spreading volume of enzyme, 150 lL, due to the larger trough area. The high content of tryptophan, 39 residues, and tyrosine, 31 residues, present in the amino acid content of the monomer give b-galactosidase a large molar extinction coefficient and make it ideal to analyze spectroscopically in situ. Analysis of the UV–Vis spectrum of b-galactosidase at the air– water interface as it was compressed is seen in Fig. 5. An inset plot of the absorbance at kmax = 276 nm displays the increase of absorbance upon compression. It is worth noting that this absorbance peak from the Langmuir monolayer is blue-shifted compared that at 281 nm in bulk solution. The observed shifting could be due to
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Wavelength (nm) Fig. 5. UV–Vis absorption at the air–water interface of the b-galactosidase Langmuir monolayer from 2 10 11 mole enzyme spread on a pH 5.7, 0.1 M NaCl subphase. (Inset) Analysis of the maximum absorbance, kmax = 276 nm, as a function of increasing surface pressure.
the air–water interface which significantly reduces the freedom degrees of molecules. The linear increase in absorbance is observed as a function of the surface pressure up to 20 mN/m which is associated with the liquid-expanded region of the isotherm. As compression continues from 25 mN/m, higher absorbance is correlated with the liquid condensed region up to the collapse of the monolayer after 35 mN/m. The increase in tryptophan and tyrosine absorbance reveals that the enzyme is not solubilized into the subphase but rather retained at the interface throughout the compression of the monolayer. If the monolayer were unstable, the absorbance intensity would not steadily increase but rather reach a plateau as molecules are expelled into the subphase. In situ fluorescence spectroscopy is also dominated by the presence of large number of tryptophan residues in b-galactosidase. With an excitation of 284 nm and maximum emission intensity at 354 nm, Fig. 6 shows the emission intensity of b-galactosidase Langmuir monolayer throughout the compression by subtracting the background scattering without depositing b-galactosidase at the air–water interface. This figure demonstrates that no increase in tryptophan emission is readily observed during the gaseous phase of the monolayer until at a surface pressure higher than
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Wavelength (nm) Fig. 6. Smoothed and normalized fluorescence spectra at the air–water interface of the b-galactosidase Langmuir monolayer from 2 10 11 mole enzyme spread on a pH 5.7, 0.1 M NaCl subphase with scattering light subtracted. The inset shows the maximum emission at 354 nm as a function of increasing surface pressure.
20 mN/m when there is a sudden increase in intensity. As the monolayer transitions to the liquid-condensed phase, this emission intensity increase corresponds with observation obtained from the absorbance data at the same surface pressures.
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3.4.2. Infrared Absorption–Reflection Spectroscopy (IRRAS) Compared with UV–Vis absorption and fluorescence spectroscopy which heavily depend on the molecular packing and surface density of protein molecules (Figs. 5 and 6), IRRAS examines the actual molecular vibrations with the information of the secondary structure of the proteins. Presented in Fig. 7 are the p-polarized IRRAS spectra of amide I and II of the enzyme monolayer collected at different surface pressures during compression at a 40° angle of incidence which had a good signal-to-noise ratio. During compression, no change in band position and no disappearance of bands reflect no change in the secondary structure of the b-galactosidase. Intensification of signal is solely due to the increase in surface density of enzyme molecules as the surface pressure decreases. The sensitivity of the spectra obtained using p-polarized light made it ideal for assigning band positions to the secondary structure and group vibrations. Band positions in the
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Wavenumber (cm-1) Fig. 7. p-Polarized infrared-reflection absorption spectroscopy of the b-galactosidase Langmuir monolayer on a pH 5.7, 0.1 M NaCl at a 40° incident angle with increasing surface pressures.
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amide I region (1700–1600 cm 1, Fig. 7) demonstrate that the b-sheet conformation dominates the secondary structure. The peaks at 1693 and 1624 cm 1 are assigned to the antiparallel b-sheet and 1670 cm 1 to the parallel b-sheet [37,38]. The amide II region (1600–1500 cm 1) shows the a-helical and random coiled content at band positions of 1555 and 1537 cm 1 as well as the b-sheet conformation at 1519 cm 1 (Fig. 7). The abundance of b-sheet content in the IRRAS spectra is expected for this enzyme due its crystal structure [39,40]. A recent study on the secondary structure of Kluyveromyces lactis b-galactosidase also found by circular dichroism that it has 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix [41]. It is worth noting that we have also studied the IRRAS spectra of the b-galactosidase Langmuir monolayer on the subphase of pH 2.0, 3.0, 4.0 and 7.0 with 0.1 M NaCl. The spectra are very similar to Fig. 7 (data not shown), indicating that the enzyme keeps its secondary structures under these conditions. 4. Conclusions In summary, b-galactosidase reproducibly forms a stable Langmuir monolayer resulting in a limiting molecular area of 42,000 Å2 molecule 1 with no formation of aggregates and little hysteresis into the NaCl subphase. In situ photophysical properties of the monolayer utilize the high content of tryptophan residues in the enzyme to confirm, at a surface pressure of 20 mN/m, that a closely packed monolayer begins to form by observing the increase in absorbance and fluorescence intensity of emission. The monolayer expands when spread on a subphase with a very acidic pH and does not form a stable monolayer when the pH rises beyond eight. p-Polarized infrared absorption–reflection spectroscopy spectra of the b-galactosidase Langmuir monolayer demonstrate mainly b-sheet content and no change in the secondary structure during compression. Notes The authors declare no competing financial interests. Acknowledgments This work is supported by the European Union’s Seventh Framework Programme, HighGlycan (contract No. 278535). R.M.L. is also grateful for the financial support from the National Science Foundation under Grant No. 1355317. References [1] J.C. Gebler, R. Aebersold, S.G. Withers, J. Biol. Chem. 267 (1992) 11126–11130. [2] U. Ohto, K. Usui, T. Ochi, K. Yuki, Y. Satow, T. Shimizu, J. Biol. Chem. 287 (2012) 1801–1812. [3] D.H. Juers, R.H. Jacobson, D. Wigley, X.-J. Zhang, R.E. Huber, D.E. Tronrud, B.W. Matthews, Protein Sci. 9 (2000) 1685–1699. [4] J.D. McCarter, D.L. Burgoyne, S. Miao, S. Zhang, J.W. Callahan, S.G. Withers, J. Biol. Chem. 272 (1997) 396–400. [5] T.-T. Nguyen, H.A. Nguyen, S.L. Arreola, G. Mlynek, K. Djinovic´-Carugo, G. Mathiesen, T.-H. Nguyen, D. Haltrich, J. Agric. Food Chem. 60 (2012) 1713– 1721. [6] J.M. Sanchez, M.A. Perillo, Biophys. Chem. 99 (2002) 281–295. [7] J.X. Feliu, A. Villaverde, FEBS Lett. 434 (1998) 23–27. [8] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, Science 285 (1999) 1569– 1572. [9] O. Renkonen, L. Penttilä, A. Makkonen, R. Niemelä, A. Leppänen, J. Helin, A. Vainio, Glycoconjugate J. 6 (1989) 129–140. [10] J.M. Sánchez, V. Nolan, M.A. Perillo, Colloids Surf. B 108 (2013) 1–7. [11] S. Taylor, B. Desbat, D. Blaudez, S. Jacobi, L. Chi, H. Fuchs, G. Schwarz, Biophys. Chem. 87 (2000) 63–72. [12] N.F. Crawford, R.M. Leblanc, Adv. Colloid Interface Sci. 207 (2014) 131–138. [13] É. Kiss, K. Dravetzky, K. Hill, E. Kutnyánszky, A. Varga, J. Colloid. Interf. Sci. 325 (2008) 337–345.
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Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Surface chemistry and spectroscopy of the b-galactosidase Langmuir monolayer Nicholas F. Crawford a, Miodrag Micic b,c, Jhony Orbulescu b, Daniel Weissbart d, Roger M. Leblanc a,⇑ a
Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, United States MP Biomedicals LLC, 3 Hutton Center, Santa Ana, CA 92707, United States c Department of Engineering Design Technology, Cerritos College, 11110 Alondra Boulevard, Norwalk, CA 92650, United States d MP Biomedicals SAS, Parc d’innovation-Rue Geiler de Kaysersberg, Illkirch-Graffenstaden 67402, France b
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history: Received 25 February 2015 Accepted 29 April 2015 Available online 7 May 2015 Keywords: b-Galactosidase Langmuir monolayer Spectroscopy Photophysical properties
a b s t r a c t The changes of interfacial properties of b-galactosidase introduced into different pH environments are investigated through surface chemistry and in situ spectroscopy. Conditions for an optimal Langmuir monolayer formation were firstly obtained by varying the subphase salt concentration and the surface-pressure area isotherm was used to extrapolate the limiting molecular area of the enzyme monolayer to be around 42,000 Å2 molecule 1. Surface pressure stability measurements held at 20 mN/m for 90 min along with compression–decompression cycles revealed no aggregate formation at the air–water interface. Consistent with the data obtained from the isotherm, in situ UV–Vis and fluorescence spectroscopy shows a steep rise in absorbance and photoluminescence intensity correlating to with a switch from a liquid-expanded to a liquid-condensed phase. A decrease in subphase pH increased the electrostatic repulsion as the enzyme was protonated, leading to an expanded monolayer. Infrared absorption–reflection spectroscopy demonstrates that the enzyme adopts mainly b-sheet conformation at the air–water interface before and during the compression. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
⇑ Corresponding author. E-mail address: [email protected] (R.M. Leblanc). http://dx.doi.org/10.1016/j.jcis.2015.04.063 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.
b-Galactosidase (E.C. 3.2.1.23) is an important family of hydrolase exoglycosidase enzymes [1,2]. Its primary function is to cleave b-glycosidic bond formed between a galactose and organic
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substrate [3,4]. To a lesser extent, this enzyme can also act on the arabinosidic and fucosidic bonds with other organic residues, such as amino acids, and it is an essential enzyme for the metabolism of both eukaryotic and prokaryotic cells. Besides fundamental interests in its role in metabolism, b-galactosidase is important tool in biochemistry, life science research and industrial biotechnology [5,6]. Its action on the X-gal substrate results in cleaving the glycosidic linkage to the indigo type dye, creating a strong indigo blue color which is a base reporting system for many ELISA assays as well as for histochemical staining [7,8]. It is also important during gene expression as a reporter gene and is the basis of the blue– white screen in molecular cloning experiments. Furthermore, its large emerging applications are in the field of glycomics, where it is used extensively in sample preparation for glycan cleavage [9]. In this paper we explore the biophysical properties of b-galactosidase at the air–water interface. A large biomacromolecule such as b-galactosidase is interesting not only for its role in biological systems as a hydrolyzing enzyme but also for its behavioral characterization which has been previously uncharacterized in a two-dimensional environment [10,11]. The Langmuir monolayer approach has been used extensively in the past to study proteins [12–14], lipids [15,16], polymers [13,17] and nanoparticles [18,19] at the interfaces, as well as to understand their structural behaviors with different environmental conditions in two dimensions. Langmuir monolayer technique has applications of simulating a hydrophobic–hydrophilic cellulous environment to investigate biomolecules at the interfaces [20,21]. The major advantages of this technique are the ease of controlling both the intermolecular interaction and ordering of the analyte via controllable variables, such as subphase and monolayer component, pH, surface pressure, surface potential and temperature [22]. Among the advantages, an important parameter easily controlled is the surface pressure that is exerted by the monolayer. When simulating a biological membrane higher surface pressures are required to accurately analyze the state of the analyte as it is compressed from an expanded phase with little intermolecular interaction to a condensed phase where high amounts of intermolecular interactions and changes in orientation are observed [23,24]. Surface-pressure area (p–A) isotherms are an ideal method of analyzing the compression of a monolayer formed by molecules such as b-galactosidase as it reduces the surface tension of the liquid subphase contained in a Langmuir trough system. Once a molecule is deposited at the air–water interface, immediate repulsion– expansion effects occur and the force energy per unit area of the subphase is lowered. As barriers skim the surface of the subphase, compressing the molecules closer together and reducing their orientation degrees of freedom, reorganization and reorientation stabilize the monolayer [23]. The force per unit length is then represented as a positive difference between the surface tension of the subphase, which should not increase in surface pressure during compression, and the increase in surface tension of the b-galactosidase monolayer until the monolayer collapses [25]. In addition to measurements involving surface-pressure, surface-potential area isotherms are performed which detail the difference in potential between the molecules at the interface and a vibrating electrode. In conjunction, these two methods reveal the state of the molecules starting at points of zero interaction and compressing them to form a condensed film and finally a compact monolayer. The heterogeneity of the monolayer is described by comparing the two isotherms which show at certain points of compression, the molecules exhibit a greater degree of electrostatic interaction between themselves and also with the subphase. Those interactions too minute to observe with physical movements such as dipole moment changes and van der Waals forces can therefore be detected.
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Not only are the physical interactions observable but also in the case of Langmuir monolayers, the photophysical properties can be determined as the molecules move to a restricted packing structure at the interface [26,27]. b-Galactosidase has a high content of tryptophan and tyrosine making it ideal to study by utilizing two in situ spectroscopic methods: UV–Vis and fluorescence spectroscopy. The high tryptophan and tyrosine content of b-galactosidase is expected to absorb incident light and produce an intensity of emission throughout these experiments [28,29]. In this article, the Langmuir monolayer technique has been implemented to analyze the surface chemistry and in situ spectroscopy of b-galactosidase during monolayer compression. Experimentation of the stability has revealed the influence of pH on the nature of the enzyme while at the air–water interface.
2. Materials and methods 2.1. Materials b-galactosidase was obtained from MP Biomedical (Solon, OH) with a monomer molecular weight of 116,646 Da as determined by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometer. The specific activity of the enzyme (735 units/mg dry weight) was provided by the company by analysis with o-nitrophenyl-b-D-galactopyranoside (ONPG) which defines one unit of the enzyme which hydrolyzes 1 lmole of the substrate per minute at 25 °C, pH 7.5. Hydrochloric acid and sodium chloride with a purity higher than 99.5%, were also obtained from MP Biomedical. Sodium hydroxide used for adjusting pH was obtained from Pharmco (Brookfield, CT). All chemicals were used without any further purification. Water utilized in these experiments was obtained from a Modulab 2020 Water purification system (Continental Water System Corp. San Antonio, TX) with resistivity of 18 MX cm, surface tension of 71.6 mN/m, and pH 5.7 at 20.0 ± 0.5 °C.
2.2. Methods Langmuir isotherms and in situ UV–Vis absorption and fluorescence spectroscopy were conducted in a clean room (class 1000) with constant temperature of 20.0 ± 0.5 °C and humidity of 50 ± 1%. A Kibron l-trough (Kibron Inc., Helsinki, Finland) with an area of 5.9 cm 21.1 cm was utilized for the surface pressure– and surface potential–area isotherms, compression–decompres sion cycles, and stability studies. The Wilhelmy method was utilized to measure surface pressure with a 0.51 mm diameter alloy wire probe with a sensitivity of ±0.01 mN/m. Surface potential was measured with a Kelvin probe consisting of a capacitor-like system. The vibrating plate was set at 1 mm above the surface of the Langmuir monolayer and a gold-plated trough acted as a counter electrode. UV–Vis absorption implemented at the air–water interface of the b-galactosidase Langmuir monolayers were obtained with a Hewlett–Packard 8452A spectrophotometer. Fluorescence spectra for b-galactosidase Langmuir monolayers were measured with an excitation wavelength kexc = 284 nm by an optical fiber detector connected to a Fluorolog-3 spectrofluorometer (Horiba Scientific, Edison, NJ) and excitation/emission both had slit widths set to 3 nm. Analysis was conducted using the maximum emission wavelength of the enzyme produced by the tryptophan moiety, kem = 354 nm. Both UV–Vis absorption and fluorescence spectra were collected on the top of a KSV trough (KSV Instrument Ltd., Helsinki, Finland), which has an area of 7.5 cm 30 cm and a quartz window located in the center of the trough. Photophysical
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3. Results and discussion 3.1. Surface pressure– and surface potential–area isotherms The interfacial properties and Langmuir monolayer behavior of b-galactosidase were investigated to optimize conditions keeping the enzyme at the air–water interface and producing a stable monolayer. Investigations of subphase salt concentration as well as spreading volume resulted in Langmuir monolayer formation with an enzyme concentration of 0.2 mg/mL and a spreading volume of 65 lL on a pH 5.7, 0.1 M NaCl subphase. Conducted in triplicate, these conditions reproducibly demonstrated all necessary phases of a proper formation of a two-dimensional enzyme Langmuir monolayer. Fig. 1 shows combined surface pressure– and surface potential– area isotherms. The surface pressure–area isotherm calculated to a spreading amount of 2 10 11 mol of enzyme at the air–water interface, a small amount which is necessary due to the large molecular weight of the molecule. An initial zero in terms of surface pressure starting at 80,000 Å2 molecule 1 is ideal correlating to a gaseous (G) phase where the floating film at the hydrophobic–hydrophilic interface has little to no intermolecular interactions [31]. Compression of the molecules at the surface results in an increase in surface pressure of the expanded film as described by a decrease in area per molecule. The liquid-expanded (L-E) phase of the monolayer starting to occur at around 65,000 Å2 molecule 1 is of interest because it shows that the molecules’ degrees of freedom are being reduced as their orientation becomes uniform. The enzyme molecules continue to condense until 30,000 Å2 molecule 1 when a steep increase in surface pressure is observed referred to as the liquid-condensed (L-C) phase where the homogenous monolayer is analogous to a three-dimensional
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spectra taken of the b-galactosidase aqueous solutions were measured by a Shimadzu UV2600 spectrophotometer recording spectra between 800 and 200 nm and a Fluorolog-3 spectrofluorometer with a slit width of 3 nm for the excitation/emission spectra, respectively. The infrared absorption–reflection spectroscopy (IRRAS) measurements of the enzyme Langmuir monolayer at the air-aqueous interface were characterized using an EQUINOX 55 Fourier transform infrared spectrometer (Bruker Optics, MA) which was connected with an XA-511 external reflection accessory equipped a mercury–cadmium–telluride (MCT) detector. The studies of IRRAS were performed using p-polarized light on a Kibron l-trough S. Each spectrum was acquired by the co-addition of 1200 scans with a resolution of 8 cm 1. UV–Vis absorption was used to analyze and confirm the concentration of the b-galactosidase bulk solution. The concentration was accurately calculated based on dilutions of the enzyme, initially a dry powder, kept at 4 °C by utilizing the reported E1% (20.9) of the enzyme [30]. Langmuir monolayers were obtained with a bulk solution concentration of 0.2 mg/mL relating to an absorbance of 0.4 at the maximum absorption of tryptophan moiety, kmax = 281 nm. Reproducible monolayers were obtained on a sodium chloride subphase (0.1 M) and the spreading volume of the enzyme was 65 lL while using the Kibron instrument and 150 lL while using the KSV instrument. At the air–water interface, deposition of the enzyme in solution was conducted by placing droplets of equal size on the surface of the subphase using a 100 lL syringe (Hamilton Co., Reno, Nevada). Experiments were then continued after a waiting time period of 15 min in order to allow the b-galactosidase monolayer to reach equilibrium at a compression rate of 6086 Å2 molecule 1 min 1. In a typical surface pressure or potential-area isotherm experiment, the barriers usually reached the maximum compression area.
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liquid. The higher surface pressure regions are of importance because of the high tension, 30–35 mN/m, found in biological membranes where molecules have the least amount of compressibility [23,32]. The final phase observed in a well-ordered monolayer is occurring around 22,000 Å2 molecule 1 where the maximum compression is reached. Extrapolating the surface pressure–area curve during the steepest part of the isotherm, typically during the liquid-condensed phase (dash line in Fig. 1), down to a zero surface pressure allows to determine the limiting molecular area [33]. The Langmuir monolayer formed from b-galactosidase has a limiting molecular area of 42,000 (±3400) Å2 molecule 1. Considering the three-dimensional crystal structure of beta-galactosidase from E. coli. (PDB: 4V40) with 107.90 Å 207.50 Å 509.90 Å, one would expect the enzyme is likely orientated as 107.90 Å 509.90 Å at the air–water interface with the axis of 207.50 Å toward the air phase. Surface potential measures the potential or dipole moment difference above and below the Langmuir monolayer film. In conjunction with surface pressure, surface potential–area isotherms as previously discussed reveal the molecular interactions occurring before and during any type of phase change of the monolayer as seen during compression. These changes in surface-potential can be correlated with phase changes in the monolayer as the minimum cross-sectional area per molecule at the air–water interface decreases. In the case of b-galactosidase, the surface potential is most influenced by dipole moment changes throughout the compression. Fig. 1 correlates the two types of monolayer measurements by showing even during the initial gaseous phase, there is an immediate increase in surface potential at 80,000 Å2 molecule 1. A kink in surface potential is observed as the monolayer begins to move into a liquid-expanded phase and as the monolayer compression transitions into a liquid-condensed phase, again a change in surface potential is observed. At a maximum voltage of 108 mV, the surface potential corresponds to a close packing structure of the enzyme molecules at the beginning of the liquid condensed phase. In the liquid-condensed phase region, while the molecular rearrangement is happening, due to the short distance (surface pressure–area isotherm), some of the dipole–dipole interaction will cancel out therefore resulting in the overall effect that the surface potential in this region is slightly decreasing. When compared, the two isotherm methods seem slightly non-aligned which is explained by the difference in detected dipole changes compared to intermolecular interactions. Surface potential is considered more sensitive in measuring these changes since
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they occur at larger distances compared to the distance needed for intermolecular interaction [34]. 3.2. The compression–decompression cycles and stability measurements of the b-galactosidase Langmuir monolayer The capacity of a monolayer to be stable for long time periods is necessary in order to perform certain in situ experimentation therefore compression–decompression cycles are conducted which
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reveal the extent of analyte which is being lost to desorption from the interface into the subphase, also known as hysteresis [35]. Once a stable and reproducible monolayer was formed, these cycles reveal in Fig. 2A–C that when compressed to 10 mN/m there is a small hysteresis because a stable monolayer has not yet been formed at this surface pressure. Although only 4% is of the initial isotherm is lost upon comparison of the first and third cycle, 20 and 30 mN/m only show hysteresis loss of 1% and 2%, respectively. The possible reason for the smaller hysteresis loss is likely due to the larger rigidity of the Langmuir monolayer when it is compressed to the higher surface pressures. Reorganization of the molecules at the interface shows that under these experimental conditions, the salt subphase renders the b-galactosidase molecules partially insoluble and good reversibility of the isotherm shows that there is little aggregate formation at the interface as well [34,36]. The stability is obtained by compressing the monolayer to the target surface pressure and keeping the surface pressure constant. The monolayer stability is evaluated using the decrease in the surface area attributed to the dissolution of the enzyme in the liquid subphase. The excellent stability of the enzyme monolayer held at a surface pressure of 20 mN/m for over a 90 min time period in Fig. 3A shows an approximate change in area per molecule to be 18%. Ideal for future experimentation, the one-molecule-thick monolayer of b-galactosidase molecules can be analyzed at this point during compression as it is transitioning between the liquid-expanded and liquid-condensed phases. Fig. 3B shows that when holding the monolayer at a higher surface pressure of 30 mN/m, a 20% shift in area per molecule occurs over the first 30 min, after which the monolayer may collapse or lose molecules to the subphase. The instability of the monolayer at this point in compression is evident by the early collapse showing that this surface pressure is not suitable for time dependent measurements. 3.3. Effect of pH on limiting molecular area
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Mean Molecular Area (Å molecule ) Fig. 2. Compression–decompression cycles for the b-galactosidase Langmuir monolayer on a 0.1 M NaCl subphase up to (A) 10, (B) 20 and (C) 30 mN/m. The arrow in each figure shows the first to the third cycle of the compression– decompression.
The limiting molecular area of the b-galactosidase Langmuir monolayer describes the minimum cross-sectional area per molecule. This value can however change with environmental conditions, namely the subphase conditions. As the extrapolated value for the limiting molecular area is shifted due to these changes, the molecular secondary conformation can be analyzed at the air–water interface. Changes in pH can affect the conformation of an enzyme by destabilizing certain interactions between amino acids throughout the molecule. Fig. 4 shows the effect of pH on the b-galactosidase Langmuir monolayer by adjusting the subphase pH while keeping the salt concentration constant at 0.1 M NaCl. The isotherms seem to collapse at the same molecular area, but this ‘‘collapse’’ is not real due to the fact that the barriers are compressed to the maximum. The isotherm initially was obtained on a subphase of pH 5.7 which is close to the isoelectric point of the enzyme, 4.6. When the pH of the subphase is around the isoelectric point of b-galactosidase, the charges resulting from functional groups that comprise the enzyme cancel out to a neutral charge, facilitating the formation of Langmuir monolayer at the air–water interface. Decreasing the pH increases the mean molecular area of the enzyme resulting in a more expanded monolayer upon compression due to a greater degree of electrostatic repulsion. This type of repulsion is expected since the overall negative charge of the enzyme based on the amino acid composition will be protonated therefore instead of the molecules being closely packed, the molecules are more spread out at the interface. On the contrary, increasing the pH away from the isoelectric point reduces the mean molecular area (Fig. 4). This is not an unexpected result from the enzyme monolayer since the basic subphase solubilizes the
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Fig. 3. Stability measurements which shows over an extended time period if the b-galactosidase monolayer can stay at the air–water interface when compressed to higher surface pressures of (A) 20 and (B) 30 mN/m. The double arrows in the figure show the percentage of mean molecular area shift at (A) 90 min and (B) 30 min in comparison with the mean molecular area when the monolayer was initially compressed to 20 and 30 mN/m.
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enzyme and hinders monolayer formation. Studies past a subphase of pH 8 (data not shown) resulted in no isotherm being produced which shows that solubilizing effects of the subphase cause the molecules to be expelled into the subphase before any type of monolayer can be formed. 3.4. In Situ photophysical properties of the b-galactosidase Langmuir monolayer 3.4.1. UV–Vis absorption & fluorescence spectroscopy The UV–Vis absorption and fluorescence spectroscopy of the b-galactosidase Langmuir monolayer were conducted on a 0.1 M NaCl subphase with a larger spreading volume of enzyme, 150 lL, due to the larger trough area. The high content of tryptophan, 39 residues, and tyrosine, 31 residues, present in the amino acid content of the monomer give b-galactosidase a large molar extinction coefficient and make it ideal to analyze spectroscopically in situ. Analysis of the UV–Vis spectrum of b-galactosidase at the air– water interface as it was compressed is seen in Fig. 5. An inset plot of the absorbance at kmax = 276 nm displays the increase of absorbance upon compression. It is worth noting that this absorbance peak from the Langmuir monolayer is blue-shifted compared that at 281 nm in bulk solution. The observed shifting could be due to
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Wavelength (nm) Fig. 5. UV–Vis absorption at the air–water interface of the b-galactosidase Langmuir monolayer from 2 10 11 mole enzyme spread on a pH 5.7, 0.1 M NaCl subphase. (Inset) Analysis of the maximum absorbance, kmax = 276 nm, as a function of increasing surface pressure.
the air–water interface which significantly reduces the freedom degrees of molecules. The linear increase in absorbance is observed as a function of the surface pressure up to 20 mN/m which is associated with the liquid-expanded region of the isotherm. As compression continues from 25 mN/m, higher absorbance is correlated with the liquid condensed region up to the collapse of the monolayer after 35 mN/m. The increase in tryptophan and tyrosine absorbance reveals that the enzyme is not solubilized into the subphase but rather retained at the interface throughout the compression of the monolayer. If the monolayer were unstable, the absorbance intensity would not steadily increase but rather reach a plateau as molecules are expelled into the subphase. In situ fluorescence spectroscopy is also dominated by the presence of large number of tryptophan residues in b-galactosidase. With an excitation of 284 nm and maximum emission intensity at 354 nm, Fig. 6 shows the emission intensity of b-galactosidase Langmuir monolayer throughout the compression by subtracting the background scattering without depositing b-galactosidase at the air–water interface. This figure demonstrates that no increase in tryptophan emission is readily observed during the gaseous phase of the monolayer until at a surface pressure higher than
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20 mN/m when there is a sudden increase in intensity. As the monolayer transitions to the liquid-condensed phase, this emission intensity increase corresponds with observation obtained from the absorbance data at the same surface pressures.
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3.4.2. Infrared Absorption–Reflection Spectroscopy (IRRAS) Compared with UV–Vis absorption and fluorescence spectroscopy which heavily depend on the molecular packing and surface density of protein molecules (Figs. 5 and 6), IRRAS examines the actual molecular vibrations with the information of the secondary structure of the proteins. Presented in Fig. 7 are the p-polarized IRRAS spectra of amide I and II of the enzyme monolayer collected at different surface pressures during compression at a 40° angle of incidence which had a good signal-to-noise ratio. During compression, no change in band position and no disappearance of bands reflect no change in the secondary structure of the b-galactosidase. Intensification of signal is solely due to the increase in surface density of enzyme molecules as the surface pressure decreases. The sensitivity of the spectra obtained using p-polarized light made it ideal for assigning band positions to the secondary structure and group vibrations. Band positions in the
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Wavenumber (cm-1) Fig. 7. p-Polarized infrared-reflection absorption spectroscopy of the b-galactosidase Langmuir monolayer on a pH 5.7, 0.1 M NaCl at a 40° incident angle with increasing surface pressures.
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amide I region (1700–1600 cm 1, Fig. 7) demonstrate that the b-sheet conformation dominates the secondary structure. The peaks at 1693 and 1624 cm 1 are assigned to the antiparallel b-sheet and 1670 cm 1 to the parallel b-sheet [37,38]. The amide II region (1600–1500 cm 1) shows the a-helical and random coiled content at band positions of 1555 and 1537 cm 1 as well as the b-sheet conformation at 1519 cm 1 (Fig. 7). The abundance of b-sheet content in the IRRAS spectra is expected for this enzyme due its crystal structure [39,40]. A recent study on the secondary structure of Kluyveromyces lactis b-galactosidase also found by circular dichroism that it has 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix [41]. It is worth noting that we have also studied the IRRAS spectra of the b-galactosidase Langmuir monolayer on the subphase of pH 2.0, 3.0, 4.0 and 7.0 with 0.1 M NaCl. The spectra are very similar to Fig. 7 (data not shown), indicating that the enzyme keeps its secondary structures under these conditions. 4. Conclusions In summary, b-galactosidase reproducibly forms a stable Langmuir monolayer resulting in a limiting molecular area of 42,000 Å2 molecule 1 with no formation of aggregates and little hysteresis into the NaCl subphase. In situ photophysical properties of the monolayer utilize the high content of tryptophan residues in the enzyme to confirm, at a surface pressure of 20 mN/m, that a closely packed monolayer begins to form by observing the increase in absorbance and fluorescence intensity of emission. The monolayer expands when spread on a subphase with a very acidic pH and does not form a stable monolayer when the pH rises beyond eight. p-Polarized infrared absorption–reflection spectroscopy spectra of the b-galactosidase Langmuir monolayer demonstrate mainly b-sheet content and no change in the secondary structure during compression. Notes The authors declare no competing financial interests. Acknowledgments This work is supported by the European Union’s Seventh Framework Programme, HighGlycan (contract No. 278535). R.M.L. is also grateful for the financial support from the National Science Foundation under Grant No. 1355317. References [1] J.C. Gebler, R. Aebersold, S.G. Withers, J. Biol. Chem. 267 (1992) 11126–11130. [2] U. Ohto, K. Usui, T. Ochi, K. Yuki, Y. Satow, T. Shimizu, J. Biol. Chem. 287 (2012) 1801–1812. [3] D.H. Juers, R.H. Jacobson, D. Wigley, X.-J. Zhang, R.E. Huber, D.E. Tronrud, B.W. Matthews, Protein Sci. 9 (2000) 1685–1699. [4] J.D. McCarter, D.L. Burgoyne, S. Miao, S. Zhang, J.W. Callahan, S.G. Withers, J. Biol. Chem. 272 (1997) 396–400. [5] T.-T. Nguyen, H.A. Nguyen, S.L. Arreola, G. Mlynek, K. Djinovic´-Carugo, G. Mathiesen, T.-H. Nguyen, D. Haltrich, J. Agric. Food Chem. 60 (2012) 1713– 1721. [6] J.M. Sanchez, M.A. Perillo, Biophys. Chem. 99 (2002) 281–295. [7] J.X. Feliu, A. Villaverde, FEBS Lett. 434 (1998) 23–27. [8] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, Science 285 (1999) 1569– 1572. [9] O. Renkonen, L. Penttilä, A. Makkonen, R. Niemelä, A. Leppänen, J. Helin, A. Vainio, Glycoconjugate J. 6 (1989) 129–140. [10] J.M. Sánchez, V. Nolan, M.A. Perillo, Colloids Surf. B 108 (2013) 1–7. [11] S. Taylor, B. Desbat, D. Blaudez, S. Jacobi, L. Chi, H. Fuchs, G. Schwarz, Biophys. Chem. 87 (2000) 63–72. [12] N.F. Crawford, R.M. Leblanc, Adv. Colloid Interface Sci. 207 (2014) 131–138. [13] É. Kiss, K. Dravetzky, K. Hill, E. Kutnyánszky, A. Varga, J. Colloid. Interf. Sci. 325 (2008) 337–345.
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